US10179921B2 - Method for producing polyphenol compounds - Google Patents

Method for producing polyphenol compounds Download PDF

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US10179921B2
US10179921B2 US14/348,490 US201214348490A US10179921B2 US 10179921 B2 US10179921 B2 US 10179921B2 US 201214348490 A US201214348490 A US 201214348490A US 10179921 B2 US10179921 B2 US 10179921B2
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coa
sequence
malonyl
phloroglucinol
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US20140315269A1 (en
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Ludovic Delage
Laurence Meslet-Cladiere
Philippe Potin
Sophie Goulitquer
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Centre National de la Recherche Scientifique CNRS
Universite Pierre et Marie Curie
Univerdite de Bretagne Occidentale
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01094—6-Deoxyerythronolide-B synthase (2.3.1.94)

Definitions

  • the present invention relates to a method for producing polyphenol compounds, i.e. phloroglucinol or one of its derivatives, with a polyketide synthase of type III (PKSIII) from a brown marine alga.
  • the invention also relates to recombinant nucleic acids coding for a polyketide synthase of type III (PKSIII) of the brown alga Ectocarpus siliculosus ( E. siliculosus ), to recombinant vectors comprising these nucleic acids, as well as to host cells comprising these vectors.
  • the invention relates to a method for preparing various compounds by means of polyphenol compounds produced according to the aforementioned method.
  • phloroglucinol and its derivatives have shown potentially interesting activities for humans in pharmacology (production of reaction intermediates for hemi-synthesis of chemical compounds), in medicine (antimicrobial, anti-HIV, anti-cancer, anti-diabetic, anti-allergic, anti-inflammatory properties) or cosmetics (anti-age effect) which makes them natural molecules with a very interesting potential (Singh I P et al., 2009, Expert Opin Ther Pat, 19: 847-66, for a review).
  • phloroglucinol synthesized chemically is marketed as a musculotropic antispasmodic drug (under the name of Spasfon® in France).
  • mixtures of products are also marketed like extracts of Ascophyllum nodosum by the Algues & Mer Ouelich corporation (29, France), products like SeanolTM, extracts of Ecklonia cava or further HealSea, extracts of Fucus vesiculosus by the Diana Naturals corporation (35, France) and a few other companies worldwide.
  • One of the present major challenges is therefore to provide methods for producing phloroglucinol or its derivatives, which are effective and rapid, and which give the possibility of obtaining these compounds in large proportions.
  • One of the possibilities consists of producing it via a biosynthesis route applying the use of enzymes.
  • PKS III polyketide synthases of type III
  • the corresponding PKS III chalcone synthases, stilbene synthases, pyrone synthases . . .
  • the inventors have identified a novel polyketide synthase in the brown marine alga Ectocarpus siliculosus, which was called PKS1.
  • PKS III polyketide synthase of type III
  • the inventors have produced a polyketide synthase of type III (PKS III) of a brown marine alga in a recombinant way and active in a heterologous system (the bacterium Escherichia coli ) with an established homogeneity purification procedure allowing production of about 5 to 10 mg of pure protein per liter of culture.
  • PKS1 was capable of synthesizing in vitro phloroglucinol from the malonyl-coenzyme A (malonylCoA).
  • the present invention relates to an isolated nucleic acid comprising or consisting in the sequence SEQ ID NO: 2 or SEQ ID NO: 4, or in a sequence at least 85% identical with the sequence SEQ ID NO: 2 or SEQ ID NO: 4, or in a complementary sequence of SEQ ID NO: 2 or of SEQ ID NO: 4.
  • said isolated nucleic acid codes for a polyketide synthase of type III.
  • the present invention additionally relates to an isolated nucleic acid coding for a polyketide synthase of type III (PKS III) comprising or consisting in:
  • FIG. 1 represents the expression vector pQE-80L (Qiagen) containing the promoter of the bacteriophage T5 inducible by isopropyl-b-D-thiogalactopyranoside (IPTG) used for the expression of the PKS1 protein.
  • pQE-80L Qiagen
  • IPTG isopropyl-b-D-thiogalactopyranoside
  • FIG. 2 represents the mass spectrometry analysis of the purified recombinant protein PKS1 of E. siliculosus.
  • FIG. 3 represents the thin layer chromatography analysis of the products formed during the enzymatic reaction using the following different substrates: malonyl-CoA alone, malonyl-CoA+acetyl-CoA, malonylCoA+hexanoyl-CoA, malonyl-CoA+lauroyl-CoA, malonyl-CoA+palmitoyl-CoA and malonyl-CoA+decanoyl-CoA.
  • FIG. 4 represents the GC-MS mass spectrometry analysis of the products formed during the enzymatic reaction using the following different substrates: malonyl-CoA alone, malonyl-CoA+acetyl-CoA, malonylCoA+hexanoyl-CoA, malonyl-CoA+lauroyl-CoA, malonyl-CoA+palmitoyl-CoA and malonyl-CoA+decanoyl-CoA.
  • FIG. 5 represents the GC-MS mass spectrometry analysis of the products formed during the enzymatic reaction using the following different substrates: malonyl-CoA alone, malonyl-CoA+acetyl-CoA, malonyl-CoA+hexanoyl-CoA, malonyl-CoA+lauroyl-CoA, malonyl-CoA+palmitoyl-CoA and malonyl-CoA+decanoyl-CoA.
  • FIG. 6 represents the LC-MS mass spectrometry analysis of the products formed during the enzymatic reaction by using malonyl-CoA alone or malonyl-CoA+acetyl-CoA.
  • 1 Negative control
  • 2 Denatured protein
  • 3 Malonyl-CoA
  • 4 Acetyl-CoA
  • 5 Lauroyl-CoA
  • 6 Palmitoyl-CoA
  • 7 Hexanoyl-CoA
  • 8 Decanoyl-CoA.
  • NL parameters 2.10 E 4, m/z: 307.1877-307.1939, MS bl.
  • FIG. 7 represents the LC-MS mass spectrometry analysis of the products formed during the enzymatic reaction by using as a substrate malonyl-CoA and lauroyl-CoA.
  • 1 Negative control
  • 2 Denatured protein
  • 3 Malonyl-CoA
  • 4 Acetyl-CoA
  • 5 Lauroyl-CoA
  • 6 Palmitoyl-CoA
  • 7 Hexanoyl-CoA
  • 8 Decanoyl-CoA.
  • ⁇ isolated>> is meant a compound which has been isolated from a living organism, such as an alga, or an animal, and/or a library of compounds.
  • the isolated nucleic acid stems from a brown marine alga.
  • the isolated nucleic acid stems from the brown marine alga Ectocarpus siliculosus ( E. siliculosus ).
  • the nucleic acids according to the invention are recombinant nucleic acids.
  • ⁇ recombinant nucleic acid>> is meant a nucleic acid, i.e. a DNA or RNA molecule, which has been subject to a biological molecular manipulation.
  • RNA molecules phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine or deoxycytidine; “DNA molecules”) in a monocatenary form or in the form of a bicatenary helix. DNA-DNA, DNA-RNA and RNA-RNA bicatenary helices are possible.
  • nucleic acid and in particular of DNA or RNA molecule only refers to the primary or secondary structure of the molecule, and is by no means limited to particular tertiary forms.
  • this term comprises bicatenary DNA which inter alia is found in linear or circular DNA molecules (for example restriction fragments), viruses, plasmids and chromosomes.
  • the sequences may be described here according to the normal convention which only gives a sequence in the direction of 5′ to 3′ along the non-transcribed strand of the DNA (i.e. the strand having a homologous sequence to mRNA).
  • sequence at least 85% identical with a reference sequence is meant that the sequence is identical to the reference sequence, except that the sequence may include up to 15 alterations of nucleotides every 100 nucleotides of the reference sequence. In other words, in order to obtain a sequence at least 85% identical with the reference sequence, up to 15% of the nucleotides of the sequence may be inserted, deleted or substituted with another nucleotide.
  • the identity percentage may be computed by producing an overall pair wise alignment based on the Needleman-Wunsch alignment algorithm for finding the optimum alignment (including “holes” or “gaps”) between two sequences over the whole of their length, for example by using Needle, and by using the BLOSUM62 matrix with a penalty for inserting ⁇ gaps>> of 10 and a penalty of extension of ⁇ gaps>> of 0.5.
  • the nucleotide sequence of the invention is at least 90%, 91%, 92%, 93%, 94%, 95%, or 99% identical with the sequence SEQ ID NO: 2 or SEQ ID NO: 4.
  • sequence differing from the sequences a) to c) by degeneration of the code>> is meant a sequence which differs from the reference sequence by a conservative substitution as indicated in the table below.
  • a nucleic acid molecule “may hybridize” to another nucleic acid molecule such as a DNA, a genomic DNA or an RNA, when the monocatenary shape of the nucleic acid molecule may hybridize with the other nucleic acid molecule under suitable conditions of temperature and of ionic force of the solution (see Sambrook et al., supra).
  • the temperature and ionic force conditions determine the “stringency” of the hybridization.
  • Low stringency hybridization conditions correspond to a T m of 55° C., for example SSC 5 ⁇ , SDS 0.1%, milk 0.25%, and no formamide; or 30% formamide, SSC 5 ⁇ , 0.5% SDS).
  • Moderate stringency hybridization conditions correspond to a higher T m (about 60° C.), for example 40% formamide, with SSC 5 ⁇ or 6 ⁇ .
  • High stringency hybridization conditions correspond to the highest T m (greater than or equal to about 65° C.), for example 50% formamide, SSC 5 ⁇ or 6 ⁇ .
  • Hybridization requires that both nucleic acid molecules contain complementary sequences, although depending on the stringency of the hybridization, mismatches between the bases are possible.
  • the suitable stringency for hybridization of the nucleic acid molecules depends on the length of the nucleic acid molecules and on the complementation degree, variables well known to those skilled in the art.
  • T m The higher the degree of similarity or homology between two nucleotide sequences, the higher is the value of the T m for the hybrids of nucleic acid molecules having these sequences.
  • the relative stability (corresponding to a higher T m ) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA.
  • equations for computing T m were derived (see Sambrook et al., supra, 9.50-0.51). For hybridization with shorter nucleic acid molecules, i.e.
  • a minimum length for a nucleic acid molecule which may hybridize is of at least about 10 nucleotides; preferably, of at least about 10 nucleotides; and more preferentially, the length is of at least about 50 nucleotides; still more preferably of at least 100 nucleotides; even more preferably of at least 1,000 nucleotides.
  • specific hybridization conditions designates a T m of 55° C. and uses conditions described above.
  • the T m is equal to 60° C., in an even more preferred embodiment, the T m is equal to 65° C.
  • a DNA “coding sequence” is a bi-catenary DNA sequence which is transcribed and translated into a polypeptide in vivo when it is placed under the control of suitable regulatory sequences. The frontiers of the coding sequence are determined by a starting codon at the end 5′ (amino) and by a stopping codon for the translation at the end 3′ (a carboxy).
  • a coding sequence may include, without being limited thereto, prokaryotic sequences, DNAc stemming from eukaryotic mRNA, genomic DNA sequences from eukaryotic DNA (for example of mammals), and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and a termination sequence of the transcription will generally be located in 3′ of the coding sequence.
  • Sequences for controlling the transcription and the translation are DNA regulatory sequences, such as promoters, activators, terminators and other similar sequences, which allow expression of a coding sequence in a host cell.
  • the polyadenylation signals are control sequences.
  • a “promoter sequence” is a DNA regulatory region capable of binding the RNA polymerase in a cell and of initiating transcription of a coding sequence towards the downstream portion (direction 3′).
  • the promoter sequence is bound to its end 3′ by the site for initiating the transcription and extends upstream (direction 5′) while including the minimum number of bases or elements required for initiating transcription to detectable levels relatively to the background noise.
  • a site is found for initiating transcription (conveniently defined, for example by mapping with the nuclease S1), as well as domains for binding to proteins (consensus sequences) responsible for the binding of the RNA polymerase.
  • Eukaryotic promoters will often contain but not always, “TATA” boxes and “CAT” boxes.
  • a coding sequence is “under the control” of sequences for controlling the transcription and the translation in a cell, when the RNA polymerase transcribes the coding sequence into mRNA which is then translated into a coded protein by the coding sequence.
  • a “signal sequence” may be included before the coding sequence.
  • This sequence codes for a signal peptide, in the N-terminal position of the protein, which orders the host cell to transport the protein on the cell surface or to secrete the protein into the medium, and this signal peptide is generally selectively degraded by the cell after export.
  • the signal sequences may be found associated with diverse native proteins of prokaryotes and eukaryotes.
  • the signal sequence consists in or comprises the sequence 5′-ATGTCTTCTGCTGCGGTTGCTATGCTGGCTGACCCGACTGTCCAGATCGCTCTGGCGT GCCTGGTGGTGTCTCTCTTCGTTGTGCTGCAGTCGGTCAAAAAG-3′ (SEQ ID NO: 5).
  • the signal sequence codes for the signal peptide of sequence MSSAAVAMLADPTVQIALACLWSLFWLQSVKK (SEQ ID NO: 6).
  • a ⁇ tag sequence>> may also be included before or after the coding sequence.
  • This sequence generally codes for a repetition of histidines, in the N-terminal position of the protein, and allows purification of the protein.
  • the tag sequence is placed before the coding sequence, and codes for the sequence of six histidines SEQ ID NO: 7 (HHHHHH), the tag sequence may therefore have as a nucleotide sequence, the sequence SEQ ID NO: 8 (5′-CAYCAYCAYCAYCAYCAY-3′).
  • the tag sequence has for an amino acid sequence, the sequence MRGSHHHHHHGS (SEQ ID NO: 9).
  • the nucleotide sequence corresponding to the tag sequence coding for the sequence SEQ ID NO: 9 may be the sequence 5′-ATGCGCGGCAGCCATCATCATCATCATCATGGCAGC-3′ (SEQ ID NO: 10).
  • the nucleic acid according to the invention codes for a PKSIII capable of synthesizing phloroglucinol from malonyl-CoA alone or in combination with other substrates, for example acetyl-CoA, hexanoyl-CoA, decanoyl-CoA, lauroyl-CoA, or palmitoyl-CoA.
  • the invention also relates to a protein coded by an isolated nucleic acid according to the invention.
  • said protein is coded by a nucleic acid comprising or consisting in the sequence SEQ ID NO: 2 or SEQ ID NO: 4.
  • said protein comprises or consists in the sequence SEQ ID NO: 1 or SEQ ID NO: 3.
  • said protein comprises or consists in a sequence having at least 93% identity, preferably at least 95% identity, or still more preferentially 99% identity with the sequence SEQ ID NO: 1 or SEQ ID NO: 3.
  • said protein is a PKSIII capable of synthesizing phloroglucinol from malonyl-CoA alone or in combination with other substrates, for example acetyl-CoA, hexanoyl-CoA, decanoyl-CoA, lauroyl-CoA, or palmitoyl-CoA.
  • sequence at least 93% identical with a reference sequence is meant that the sequence is identical with the reference sequence, except that the sequence may include up to seven alterations of amino acids per every 100 amino acids of the reference sequence. In other words, in order to obtain a sequence at least 93% identical with the reference sequence, up to 7% of the amino acids of the sequence may be inserted, deleted, or substituted with another amino acid.
  • the at least 93% identical sequence is a sequence homologous to the reference sequence, i.e. it differs from the reference sequence by one or several conservative substitutions.
  • the identity percentage may be computed by producing an overall pairwise alignment based on the Needleman-Wunsch alignment algorithm for finding the optimum alignment (including “holes” or “gaps”) between two sequences over the whole of their length, for example by using Needle, and by using the BLOSUM62 matrix with a penalty for inserting ⁇ gaps>> of 10 and a penalty for extending ⁇ gaps>> of 0.5.
  • ⁇ conservative substitutions>> is meant the replacement of an amino acid with another one, without altering the conformation and the function of the protein, including, but without being limited thereto, the replacement of an amino acid with another amino acid having the same properties (such as for example, the same polarities, potentials for binding to hydrogen, acid, basic, hydrophobic, aromatic and other properties).
  • the amino acids having the same properties are known to one skilled in the art.
  • arginine, histidine and lysine are hydrophilic basic amino acids which may be interchangeable.
  • isoleucine, a hydrophilic amino acid may be replaced with leucine, methionine or valine.
  • the hydrophilic neutral amino acids which may be substituted with each other, include asparagine, glutamine, serine and threonine.
  • the present invention includes the amino acids which have been altered or modified from natural amino acids.
  • amino acids may be grouped as described by Lehninger (1975, Biochemistry, Second Edition, Worth Publishers, Inc. New-York: NY., pp. 71-77), as described in Table 2 below:
  • the invention also relates to a vector comprising a nucleic acid according to the invention, wherein the said nucleic acid is placed under the control of signals (i.e. a promoter, a terminator and/or an enhancer) allowing expression of the nucleic acid according to the invention.
  • the vector may further comprise a gene for resistance to an antibiotic, such as ampicillin or kanamycin.
  • ⁇ vector>> designates an extrachromosomal element which may bear a non-essential gene for cell metabolism, and which generally is a circular double strand DNA.
  • the extrachromosomal element may be a self-replicating sequence, a phage sequence or a nucleotide sequence, a single or double strand DNA or RNA, a plasmid, a cosmid.
  • a vector contains regulatory sequences for transcription or translation, a selection marker, or a sequence allowing self-replication or chromosomal insertion.
  • a suitable vector includes the region 5′ of a gene which regulates the initiation of the transcription (i.e. a promoter) and a region 3′ which controls the termination of the transcription (i.e.
  • the promoter may for example be that of CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, lac, trp, ⁇ P L , ⁇ P R , T7, tac, bacteriophage T5 or trc.
  • the terminator may be derived from many genes of a preferred host cell and may optionally be omitted.
  • the vector is a pQE-80L plasmid (Qiagen) containing the promoter of the T5 bacteriophage which may be induced by isopropyl- ⁇ -D-thiogalactopyranoside (IPTG).
  • the nucleic acids and/or the vector according to the invention may be used for transforming a cell or a host organism, i.e. for expressing or producing a protein according to the invention.
  • another aspect of the invention relates to a host or a host cell which contains a nucleic acid or a vector according to the invention, or which expresses (or is capable of expressing under suitable conditions) a protein according to the invention.
  • Suitable hosts and host cells are known to one skilled in the art, and may for example be any fungus, cell or eukaryotic or prokaryotic cell line, eukaryotic or prokaryotic organisms.
  • the host or the host cell may be (i) a bacterial strain, including without being limited thereto, Gram-negative bacteria lines such as lines of Escherichia, for example Escherichia coli; of Proteus, for example Proteus mirabilis; of Pseudomonas, for example Pseudomonas fluorescens; and Gram-positive bacteria strains such as lines of Bacilli, for example Bacillus subtilis or Bacillus brevis; of Streptomyces, for example Streptomyces lividans; of Staphylococcus, for example Staphylococcus carnosus; and of Lactococcus, for example Lactococcus lactis; (ii) a fungal cell, including without being limited thereto, cells of the species Trichoderma, for example Trichoderma reesei; Neurospora, for example Neurospora crassa; Sordaria, for example Sordaria macrospore; Aspergillus
  • the host cell is a bacterium of the Escherichia coli type, and more preferentially the host cell is the strain E. coli BL21-Codin Plus-RI LP (Stratagene).
  • Another aspect of the invention relates to a method for producing a polyketide synthase, comprising the steps of:
  • the method further comprises a step for transforming a host cell by means of a recombinant vector as defined earlier before step a). Accordingly, the method according to the invention may comprise the steps of:
  • the transformation in step a0) may be carried out with methods known to one skilled in the art, for example, by transfection, electroporation, electrotransfer, microinjection, transduction, merging of cells, DEAE-dextran, precipitation with calcium phosphate, lipofection, use of a gene gun, or a DNA vector carrier (see for example, Wu et al., 1992, J. Biol Chem. 267:963-967; Wu et al., 1988, J. Biol Chem. 263:14621-14624; Hartmut et al., Canadian patent application No. 2,012,311, published on Mar. 15, 1990).
  • the transformation in step a0) is carried out with a thermal shock on cells made chemically competent, in a suitable culture medium.
  • the thermal shock is carried out between 35° C. and 50° C. for 30 seconds to 1 min followed by a return into ice for 1 to 3 min. Still more preferably, the thermal shock is achieved at 42° C. for 45 seconds followed by a return into ice for 2 min.
  • suitable culture medium is meant is a culture medium allowing the growth of transformed cells.
  • a culture medium is known to one skilled in the art. For example, this is a medium including, without being limited thereto, a carbonaceous substrate or a source of carbon which may be metabolized by the transformed cell.
  • the carbonaceous substrate or the source of carbon may be selected from monosaccharides, oligosaccharides, polysaccharides, simple-carbon substrates, and a mixture of these compounds.
  • the culture medium contains an antibiotic, such as ampicillin or kanamycin.
  • the cell transformed in step a0) is a bacteria of the E. coli type such as the strain E. coli BL21-CodinPlus-RILP (Stratagene), transformed with a vector and/or a nucleic acid according to the invention, and cultivated by using a suitable medium, such as the Luria-Broth (LB) medium, preferably containing at least 100 ⁇ g/ml of ampicillin.
  • E. coli type such as the strain E. coli BL21-CodinPlus-RILP (Stratagene)
  • a suitable medium such as the Luria-Broth (LB) medium, preferably containing at least 100 ⁇ g/ml of ampicillin.
  • LB Luria-Broth
  • the culture made in step a) has the purpose of allowing expression of the recombinant polyketide synthase. It may be made by methods known to one skilled in the art, for example by means of a bio-reactor, also called a fermenter.
  • the bio-reactor is a hermetically sealed tank provided with a stirring system, a ventilation system, with probes being used for measuring various parameters (pH, temperature, dissolved oxygen) and supply pores allowing accurate dosage of the composition of the culture medium during fermentation.
  • the fermentation in the bio-reactor is carried out in two phases: a growth phase I during which the cells divide at an accelerated rate, and then an accumulation or induction phase II leading to the expression of the recombinant protein.
  • a growth phase I during which the cells divide at an accelerated rate
  • an accumulation or induction phase II leading to the expression of the recombinant protein.
  • the phases I and II may be made as described in Example 1.3.
  • the extraction and/or the purification in step b) may be carried out by means of methods known to one skilled in the art.
  • the extraction may be achieved by lysis of the cells by means of a French press, by sonication, or via an enzymatic route, or with any other standard technique.
  • the lysis of the cells may be followed by a filtration step, and/or centrifugation of the lysate.
  • the purification may be achieved by means of chromatography (on or ion exchange column affinity or size-separation), centrifugation, differential solubility or by any other standard technique.
  • the extraction and/or the purification may be achieved as described in Example 1.3.
  • said production method allows production of at least 1 mg/L to 20 mg/L of pure protein, and still preferably at least 5 to 10 mg/L of pure protein.
  • PKSIII polyketide synthase of type III extracted from a brown alga was capable of synthesizing phloroglucinol and/or one of its derivatives from diverse carbonaceous substrates, and more particularly from malonyl-CoA.
  • the present invention therefore also relates to a method for producing at least one polyphenol compound, wherein:
  • the method includes a step for recovering the phloroglucinol and/or one of its derivatives.
  • This recovery step may comprise an extraction step and/or a purification step.
  • ⁇ polyphenol compound>> is meant a compound stemming from the secondary metabolism of the plant kingdom, i.e. land plants and aquatic macrophytes, which have at least one aromatic ring with 6 carbons (phenol), itself bearing one or several hydroxyl functions (OH).
  • phenol aromatic ring with 6 carbons
  • OH hydroxyl functions
  • Many families of molecules are distinguished, for which the structure is relatively close: flavonoids (yellow-orangey plant pigments), anthocyans (compounds with red to violet colors responsible for the purple color of red grapes) and tannins.
  • ⁇ polyketide synthase of type III>> is meant an enzyme capable of synthesizing phloroglucinol and/or one of its derivatives from a carbonaceous substrate.
  • PKSIII is obtained from a brown marine alga
  • PKSIII is obtained from brown marine algae of the Ectocarpus genus
  • PKSIII stems from the brown marine alga Ectocarpus siliculosus.
  • PKSIII is produced by the method according to the invention described above.
  • PKSIII comprises or consists in a protein according to the invention. More particularly, PKSIII comprises or consists in the sequence SEQ ID NO: 1 or SEQ ID NO: 3 or a sequence having at least 93%, 95% or 99% identity with the sequence SEQ ID NO: 1 or SEQ ID NO: 3.
  • sequence having at least 93% identity with SEQ ID NO: 1 or SEQ ID NO: 3 may differ from the reference sequence (i.e. SEQ ID NO: 1 or SEQ ID NO: 3) by one or several conservative substitution(s).
  • the terms of ⁇ conservative substitutions>> are as defined above.
  • PKSIII consists in the sequence SEQ ID NO: 1 or SEQ ID NO: 3.
  • said PKSIII comprises or consists in a sequence which may be coded by the nucleotide sequence SEQ ID NO: 2 or a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 99% identity with SEQ ID NO: 2.
  • said PKSIII comprises or consists in a sequence which may be coded by the nucleotide sequence SEQ ID NO: 4 or a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 99% identity with SEQ ID NO: 4.
  • sequence having at least 85% identity with SEQ ID NO: 2 or SEQ ID NO: 4 may differ from the reference sequence (i.e. SEQ ID NO: 2 or SEQ ID NO: 4) by one or several conservative substitution(s).
  • the terms of ⁇ conservative substitutions>> are as defined above.
  • the carbonaceous substrate includes compounds of the metabolite type, such as C 1 -C 18 carbon chains, fatty acids, mono-, di-, tri-glycerides, polyols, phospholipids, phosphoacids, monosaccharides, amino acids, nucleotides, hydrolyzable homo- or hetero-oligomers or polymers of these compounds, and the biologically active forms of these compounds.
  • Said metabolites may be of any biological or synthetic origin.
  • Other examples of compounds are aromatic, aliphatic and cycloaliphatic C 1 -C 18 compounds.
  • the carbonaceous substrate is selected from malonyl-CoA, acetyl-CoA, hexanoyl-CoA, decanoyl-CoA, lauroyl-CoA, and/or palmitoyl-CoA.
  • the carbonaceous substrate is malonyl-CoA.
  • ⁇ phloroglucino>> is meant benzene-1,3,5-triol, its CAS number is 108-73-6.
  • ⁇ derivative of phloroglucinol>> are notably meant the following compounds: acetyl-phloroglucinol, lauroyl-phloroglucinol, palmitoyl-phloroglucinol, hexanoyl-phloroglucinol, decanoyl-phloroglucinol.
  • the carbonaceous substrate is malonyl-CoA and the produced phenol compound consists in or comprises phloroglucinol.
  • the carbonaceous substrate is malonyl-CoA and acetyl-CoA
  • the produced phenol compound consists in or comprises phloroglucinol and acetyl-phloroglucinol.
  • the carbonaceous substrate is malonyl-CoA and lauroyl-CoA and the produced phenol compound consists in or comprises phloroglucinol and lauroyl-phloroglucinol.
  • the carbonaceous substrate is malonyl-CoA and palmitoyl-CoA and the produced phenol compound consists in or comprises phloroglucinol and palmitoyl-phloroglucinol.
  • the carbonaceous substrate is malonyl-CoA and hexanoyl-CoA and the produced phenol compound consists in or comprises phloroglucinol and hexanoyl-phloroglucinol.
  • the carbonaceous substrate is malonyl-CoA and decanoyl-CoA and the produced phenol compound consists in or comprises phloroglucinol and decanoyl-phloroglucinol.
  • phloroglucinol or one of its derivatives By majority production of phloroglucinol or one of its derivatives, is meant that the phloroglucinol or one of its derivatives is the product which is found in the highest concentration relatively to the other produced compounds. Preferentially, the production of phloroglucinol or one of its derivatives is of at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.
  • the step for putting the polyketide synthase in contact with at least one carbonaceous substrate is known to one skilled in the art. Preferably, but in a non-limiting way, this step is carried out as described in Examples 1.4 or 1.5. More particularly, it comprises the steps of (i) putting the polyketide synthase in contact with the carbonaceous source in a suitable medium, (ii) incubating the thereby obtained mixture.
  • the suitable medium in step (i) may be a HCl-EDTA medium, for which the respective concentrations are comprised between 30 and 70 mM of HCl and 0.5 and 2 mM of EDTA, preferentially comprising between 45 and 55 mM of HCl and 0.8 and 1.2 mM of EDTA, even more preferentially the HCl and EDTA concentrations are 50 mM and 1 mM, respectively.
  • the pH of the medium is comprised between 6 and 8, and still preferably, the pH is of about 7.5.
  • the incubation (ii) is preferably carried out at room temperature, i.e. at a temperature comprised between 20° C. and 30° C., and for a duration comprised between 30 mins and 8 hrs. Still preferably, the incubation is carried out at a temperature comprised between 22° C. and 28° C., and still preferably the incubation is carried out at about 25° C. Preferably, the incubation is carried out for a duration comprised between 1 hr and 5 hrs, and still preferably the incubation is carried out for about 1 hr, 2 hrs, 3 hrs, 4 hrs or 5 hrs. In a particularly preferred embodiment, the incubation is carried out at about 25° C. for about 1 hr, 2 hrs, 3 hrs, 4 hrs or 5 hrs.
  • the extraction and/or purification step may be carried out with techniques known to one skilled in the art.
  • the extraction may be achieved by means of ethyl acetate.
  • the purification step may for example be achieved by chromatography on a thin layer (TLC) or by gas chromatography coupled with mass spectrometry (GC-MS).
  • polyphenol compounds produced by the method according to the invention such as phloroglucinol and its derivatives, may be used in many applications in the pharmaceutical, nutraceutical, cosmetic, agri-feed or plant protection fields.
  • the invention also relates to a method for producing a pharmaceutical, nutraceutical, cosmetic, agri-feed or plant protection compound, said method comprising the steps of:
  • step b) optionally modifying said phenol compound obtained in step a) for producing a pharmaceutical, nutraceutical, cosmetic, agri-feed or plant protection compound,
  • the invention also relates to the use of the polyphenol compounds produced by the method according to the invention for preparing pharmaceutical, nutraceutical, cosmetic, agri-feed or plant protection compositions.
  • the object of the invention is also these compositions.
  • the pharmaceutical or nutraceutical compositions may contain supports or the like (carriers, excipients, diluents) and/or adjuvants which are pharmaceutically acceptable.
  • supports or the like carriers, excipients, diluents
  • adjuvants which are pharmaceutically acceptable.
  • pharmaceutically acceptable preferably means approved by a government regulatory agency, in particular recommended in the American or European Pharmacopoeia, for a use in animals, and more particularly in humans.
  • Suitable pharmaceutical supports or the like are notably described in “Remington's Pharmaceutical Sciences” by E. W. Martin.
  • These pharmaceutically acceptable supports or the like may be sterile liquids, such as water and oils, including liquids derived from petroleum, animals, plants, or of synthetic origin, such as groundnut oil, soya bean oil, mineral oil, sesame oil, and other similar oils. Water is a preferred support when the pharmaceutical composition is administered via an intravenous route. Saline solutions and aqueous solutions of dextrose and glycerol may also be used as liquid supports, in particular for injectable solutions.
  • Suitable pharmaceutical excipients comprise mannitol, human serum albumin (HSA), starch, glucose, lactose, saccharose, gelatin, malt, rice, flour, chalk, silica gel, magnesium carbonate, magnesium stearate, sodium stearate, glycerol monostearate, talcum, sodium chloride, powdered skimmed milk, glycerol, propylene, glycol, water, ethanol and other similar excipients.
  • HSA human serum albumin
  • starch glucose, lactose, saccharose, gelatin, malt, rice, flour, chalk, silica gel, magnesium carbonate, magnesium stearate, sodium stearate, glycerol monostearate, talcum, sodium chloride, powdered skimmed milk, glycerol, propylene, glycol, water, ethanol and other similar excipients.
  • compositions will contain a diagnostic or therapeutic effective amount of the active compound with a suitable amount of the support or the like so as to provide a form for a suitable administration to the patient.
  • intravenous injection is a highly efficient administration form, other methods may be used, such as an injection, or nasal or parenteral administration.
  • cosmetic compositions may contain a cosmetically acceptable carrier.
  • a cosmetically acceptable carrier is meant a carrier suitable for use in contact with human and animal cells, in particular the cells of the epidermis, without any toxicity, irritation, induced allergic response or the like, and in proportion with an advantage/reasonable risk ratio.
  • a cosmetically acceptable carrier mention may notably be made of water, in particular distilled water.
  • the cosmetic compositions according to the invention may further comprise any additive customarily used in the cosmetic field, such as sequestering agents, anti-oxidants, preservatives, fillers, electrolytes, humectants, coloring agents, usual bases or acids, either mineral or organic, perfumes, essential oils, cosmetic actives, moisteners, vitamins, essential fatty acids, sphingolipids, self-tanner compounds, skin soothing and protective agents.
  • additives may be present in the composition in an amount from 0.001% to 20% by weight based on the total weight of the composition.
  • the cosmetic compositions of the invention may contain customary adjuvants in the cosmetic or dermatological field, such as emulsifiers, hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic actives, preservatives, anti-oxidants, perfumes, fillers, filters and coloring materials.
  • the amounts of these different adjuvants are those conventionally used in the cosmetic and/or dermatological fields and, for example are from 0.01% to 20% of the total weight of the composition.
  • These adjuvants depending on their nature, may be introduced into the fatty phase, into the aqueous phase and/or into the lipid vesicles.
  • the application of a cosmetic composition according to the invention is carried out via a topical route on the skin, including the mucosas, notably the lips or appendages.
  • the application of a cosmetic composition according to the invention may also be carried out by intradermal injection.
  • the cosmetic compositions according to the invention may appear in all the galenic forms normally used for topical application, in the form of an ointment, a cream, an oil, a milk, a pomade, a powder, a swab, a solution, a gel, a spray, a lotion, a suspension, a soap.
  • compositions of the invention may be cosmetic compositions in the form of an oil-in-water or water-in-oil emulsion, or a multiple emulsion, a microemulsion, a hydroalcohol gel, a cream, an oil, a hydroalcohol lotion.
  • compositions of the invention are particularly useful, for moistening, soothing, repairing and/or protecting the skin.
  • compositions are also particularly advantageous for controlling skin aging i.e. notably the phenomena of wrinkles, loss of tonicity and elasticity due to structural modifications of the skin due to aging.
  • the cosmetic compositions of the invention are also useful for protecting the skin from exterior aggressions, such as notably ultraviolet rays or air contaminations.
  • the plant protective compositions according to the invention may further comprise one or several surfactants, preservatives, dispersants, wetting agents, emulsifiers, anti-foam agents, water.
  • the plant protective compositions according to the invention may be formulated in different forms, for example in the form of wettable powders, dispersible granules, concentrated suspensions, powders for powdering.
  • the invention also relates to the use of phloroglucinol and/or one or several of its derivatives produced by the method according to the invention for treating spasmodic disorders, viral diseases, parasite diseases, microbial diseases, fungal diseases, dermatological disorders, hypertension, osteoporosis, inflammatory diseases, vascular diseases, sexual disorders, cancers, diabetes, neurodegenerative diseases, depression and allergy.
  • the invention also relates to methods for treating spasmodic disorders, viral diseases, parasite diseases, microbial diseases, fungal diseases, dermatological disorders, hypertension, osteoporosis, inflammatory diseases, vascular diseases, sexual disorders, cancers, diabetes, neurodegenerative diseases, depression and allergy, comprising the administration of phloroglucinol and/or of one or several of its derivatives to a patient in need thereof.
  • Spasmodic disorders may notably comprise functional disorders of the digestive tract (colitises) and of bile ducts, renal or hepatic colics, gynecological pains, and contractions during pregnancy.
  • Viral diseases may notably comprise infections with the acquired human immunodeficiency virus (HIV) and with the virus of herpes.
  • HIV human immunodeficiency virus
  • the parasite diseases may notably comprise malaria.
  • Microbial diseases may notably comprise infections due to Gram-negative or Gram-positive bacteria, and more particularly the bacteria of the types Streptococcus mutans, Porphyromonas gingivalis, Bacillus subtilis and Staphylococcus aureus.
  • the fungal diseases may notably comprise infections due to Aspergillus niger, Aspergillus flavus, Mucor and Cladosporium.
  • Dermatological disorders may notably comprise psoriasis, skin alopecia, skin healing problems, and skin aging i.e. notably the phenomena of wrinkles, loss of tonicity and elasticity due to structural modifications of the skin due to aging.
  • Vascular diseases may notably comprise Raynaud's disease and acrocyanosis.
  • Neurodegenerative diseases may notably comprise Alzheimer's disease, Kiloh Nevin's syndrome, carpal tunnel syndrome, Tardy Ulnar's paralysis, Guyon's canal syndrome.
  • Sexual disorders may notably comprise erection disorders.
  • Phloroglucinol or its derivatives may be used alone or in a combination with other compounds having a therapeutic activity for treating the disorders mentioned above.
  • the compounds malonyl-CoA, acetyl-CoA, hexanoyl-CoA, lauroyl-CoA, palmitoyl-CoA and decanoyl-CoA are from Sigma.
  • the [ 2-14 C] Malonyl-CoA (55 mCi/mmol) is from Perkin Elmer (USA).
  • the strain Escherichia coli DH5a [fhuA2 _(argF-lacZ)U169 phoA glnV44 ⁇ 80 _(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17] (Stratagene) was used as a host strain for maintaining plasmids.
  • the ⁇ cis-repressed>> pQE-80L derivatives were transformed into the strain E. coli BL21 (DE3) codon Plus RILP [ E.
  • the gene coding for the PKS1 of E. siliculosus was amplified by PCR (30 amplification cycles) from the cDNA described in Cock et al., 2010, Nature 465(7298): 617-621.
  • the expression vector pQE-80L (Qiagen) containing the promoter of the bacteriophage T5 inducible by isopropyl-b-D-thiogalactopyranoside (IPTG) was used for expressing the protein ( FIG. 1 ).
  • siliculosus was cloned in the vector at the restriction sites SphI and HindIII by using the oligonucleotides PQECHSFowBis (5′-GGCGGATCCGCATGCATGTCCAAGGACGAGCAGACGGTATACCCGGTCATCGCC-3′ (SEQ ID NO: 11)) and PQECHSRev (5′-GGCTAAGCTTTTACTAGATCTGCCTGAGAAGGATGCCCTCTGCCCC-3′ (SEQ ID NO: 12)).
  • the PCR conditions used for the cloning are the following: 50 ng of DNAc PKS1, 0.4 ⁇ M of each oligonucleotide, 0.4 mM of dNTP mix in a reaction medium of 50 ⁇ L with the Phusion enzyme (Finnzyme) with its HF buffer according to the recommendations of the supplier.
  • the reaction was carried out in 3 steps: denaturation of the DNA and of the primers at 98° C. for 5 min, 30 PCR cycles at 98° C. for 30 s, 52° C. for 30 s, 72° C. for 2 min and then finally an elongation end step at 72° C. for 7 min.
  • the ligation product is introduced by a thermal shock into competent cells DHSalpha prepared in the laboratory, and a transformant is selected for mass production of a recombinant vector containing the PKS1 insert.
  • the plasmid is purified with the Miniprep SV kit (Promega). After checking the nucleotide sequence of PKS1, the insert is digested by SphI and HindIII, and then purified on agarose gel with the MinElute kit (Qiagen) before being ligated into the vector pQE80L (Qiagen) doubly digested with the same restriction enzymes and dephosphorylated by SAP (NE Biolabs).
  • the ligation conditions in pQE80L are the same as described earlier.
  • the constructs code for a total protein to which a label of six histidines (His-tag) was added to its N-terminal end.
  • the constructs were transformed into the strain E. coli BL21-CodonPlus-RILP strain (Stratagene) by using a solid LB medium containing 100 ⁇ g/mL of ampicillin.
  • the expression of the recombinant protein was achieved by cultivating the bacteria in a ZYP medium at 20° C. by using a 5 L fermenter batch. After 48 hours of cultivation, induction of the protein expression is continued by adding 0.5 mM of IPTG. After this last induction, the cells are collected by centrifugation and frozen to ⁇ 80° C.
  • the cells are then re-suspended in the medium A (20 mM Tris-HCl pH 7.5, 300 mM NaCl and 50 mM Imidazole) supplemented with a mixture of a protease inhibitor, of the lysozyme (1 mg/mL) and of the DNase (10 mg/ml).
  • the lysis of the cells is then carried out with two passes into a French press in order to reduce the viscosity of the supernatant.
  • the cell debris are removed by centrifugation at 20,000 rpm at 4° C. for 1 hr 30 mins.
  • the supernatant is then transferred into an Ni-sepharose column (GE Healthcare).
  • the cell extract is then fractionated by affinity chromatography, so called ⁇ IMAC>> (Immobilized-metal affinity chromatography) on an ⁇ KTATM-Avant apparatus (GE Healthcare).
  • ⁇ IMAC>> Immobilized-metal affinity chromatography
  • GE Healthcare ⁇ KTATM-Avant apparatus
  • the proteins are eluted according to a procedure with a gradient from 50 mM to 500 mM of imidazole by mixing the buffer A with the buffer B (20 mM Tris-HCl pH 7.5; 300 mM NaCl; 500 mM imidazole).
  • fractions C12 to E10 were then concentrated to 5 ml by ultrafiltration on a 10 kDa CentriPrep (Millipore) and simultaneously exchanged in a buffer containing 20 mM of Tris-HCl, pH 7.5 100 mM NaCl.
  • the proteins were then transferred into a filtration column on a Superdex S-200 HR 16/60 gel (GE Healthcare) and purified by steric exclusion chromatography by using an ⁇ AKTA-Avant apparatus (GE Healthcare). The purity and the integrity of all the protein samples were analyzed by electrophoresis on a 12% SDS-polyacrylamide gel and by MALDI-TOF mass spectrometry.
  • the radioactive signals were detected and quantified by means of a Typhoon imaging system (Molecular Dynamics-GE Healthcare).
  • TMS-ether Trimethylsilyl-ethers
  • the metabolites were re-suspended in a 100 ⁇ l of hexane and analyzed by GC-MS in an El mode on an Agilent GC 6890 coupled with a detector ⁇ 5973 MS Detector>> (Agilent, Les Ulis, France) and equipped with a DB-5MS column (30 m ⁇ 0.25 mm of internal diameter ⁇ 0.25 ⁇ m of film thickness (J and W Scientific, Agilent)).
  • the temperatures of the injection orifice and of the interface are 250 and 280° C. respectively; that of the ion source and of the MS analyser were respectively set to 230 and 150° C.
  • the samples were injected in a ⁇ division-less injection>> mode or a ⁇ splitless>> mode.
  • the temperature of the oven was first set to 60° C. for 5 min, and then increased by 10° C./min up to 100° C., raised at a rate of 1° C./min up to 150° C. and finally raised at a rate of 8° C./min up to 290° C., and then maintained for 5 mins.
  • the compounds were ionized by impacts of electrons with an energy of 70 eV.
  • the analytes were detected by a total ion current of 50 to 850 m/z. All the data were processed with the MSDchem software package (EMBL-EBI).
  • a liquid chromatography system DionexUltiMate 3000Rapid Separation LC (RSLC, Dionex) is coupled with a hybrid mass spectrometer LTQ-OrbitrapTM (Thermo Fisher Scientific). All the solvents and reagents used are of an analytical grade or HPLC grade (Carlo Erba).
  • the chromatographic separation was achieved on a column Acclaim RSLC 120, C18, 2 ⁇ m particle size, 2.1 ⁇ 100 mm column (Dionex) maintained at 20° C.
  • the mobile phase is water containing 0.1% of acetic acid (A) and acetonitrile containing 0.1% of acetic acid (B).
  • the flow was adjusted to 0.25 ml.min ⁇ 1 .
  • the elution gradient (A:B, v/v) was produced as described in the following: 80:20 from 0 to 1 min; and then 0:100 from 1 to 10 mins; and then 20:100 for 10 mins and for 20.1 mins, 80:20 for 10 mins.
  • the injected volume is 50 ⁇ L.
  • HPLC column was connected without uncoupling with the electrospray operative interface in a negative mode.
  • the voltage of the spray was 3.5 kV and the temperature of the transfer capillary was maintained to 350° C.
  • the “sheath liquid” and the auxiliary nitrogen gas were applied for assisting evaporation of the solvent at a flow of 25 and 5 arbitrary units respectively.
  • the totality of the scans of the mass spectra was obtained for m/z from 50 to 2,000 by using a mass resolution of 30,000 FWHM at 400 m/z in a profile mode.
  • the gene PKS1 present on the genome of E. siliculosus has been available in public data bases since June 2010 and it corresponds to a sequence of 1,245 nucleotides, coding for a protein with 415 amino acids.
  • An addressing signal peptide was predicted according to the use of the SIGNALP v.2.0 software package which uses the two Neural Networks and Hidden Markov models (Nielsen et al., Protein Eng, 1999, 12: 3-9) and this sequence of 102 nucleotides, corresponding to the first 34 amino acids was removed in order to obtain a mature recombinant protein expressed in the bacterial cytoplasm.
  • the gel for electrophoresis of the elution fractions from the affinity column ⁇ IMAC>> stained with Coomassie blue allows detection of the production of a recombinant protein at the expected size of about 50 kDa.
  • a second purification on a size exclusion column gave the possibility of obtaining the recombinant protein with a homogeneity of more than 99% according to DLS (Dynamic Light Scattering) analysis.
  • the production yield for a culture in a 5 L fermenter was estimated to be about 5 to 10 mg of active recombinant protein per liter of culture and may therefore support an industrial production level. Further, the enzyme proved to be stable for one to two months at 4° C. and it supports freezing to ⁇ 80° C.
  • the analysis of the products formed in TLC indicate that there is formation of phloroglucinol with or without starters (malonyl-CoA alone, malonyl-CoA+acetyl-CoA, malonyl-CoA+hexanoyl-CoA, malonyl-CoA+lauroyl-CoA, malonyl-CoA+palmitoyl-CoA and malonyl-CoA+decanoyl-CoA) and that the main obtained compounds of the reaction are phloroglucinol in the case of malonyl-CoA alone or of malonyl-CoA+acetyl-CoA and an acyl-phloroglucinol in the case of the malonyl-CoA with the other starters (acetyl-CoA, hexanoyl-CoA, lauroyl-CoA, palmitoyl-CoA and decanoyl-CoA) ( FIG. 3 ).
  • the formation of the acyl-phloroglucinols was detected by specifically extracting the compounds present on TLC and by analyzing them with LC-MS mass spectrometry.
  • the enzyme PKS1 produces lauroyl-phloroglucinol ( FIG. 7 ).
  • PKS1 The overall structure of a PKS1 monomer reveals a composition with ⁇ helices and ⁇ sheaths organized in a canonical fold of the ⁇ -thiolase type.
  • the catalytic triad is represented by the residues Cys194, His331 and Asn364.
  • PKS18 PKS1 seems to have a tunnel for accessing the catalytic site where acyl-CoAs with more or less long chains may step in.
  • the potential binding site of malonyl-CoA was also identified.

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CN114990084B (zh) * 2022-05-13 2024-08-23 中国医学科学院药用植物研究所 白木香来源的Ⅲ型聚酮合酶AsPKS4及其编码基因和应用
FR3147291A1 (fr) 2023-03-28 2024-10-04 Compagnie Generale Des Etablissements Michelin Utilisation de polykétide synthases de type III de cyanobactéries comme phloroglucinol synthases
FR3147290A1 (fr) 2023-03-28 2024-10-04 Compagnie Generale Des Etablissements Michelin Utilisation de polykétide synthases de type III de champignons Ascomycètes comme phloroglucinol synthases
EP4541885A1 (fr) 2023-10-20 2025-04-23 Compagnie Generale Des Etablissements Michelin Synthases de phloroglucinol optimisees

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11512293B2 (en) * 2017-06-30 2022-11-29 Compagnie Generale Des Etablissements Michelin Use of type III polyketide synthases as phloroglucinol synthases
US11920178B2 (en) 2017-06-30 2024-03-05 Compagnie Generale Des Etablissements Michelin Use of type III polyketide synthases from bacteria as phloroglucinol synthases

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US20140315269A1 (en) 2014-10-23
PT2761005T (pt) 2018-10-22
FR2980801B1 (fr) 2016-02-05
FR2980801A1 (fr) 2013-04-05
EP2761005A1 (fr) 2014-08-06
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